Method of evaluating antifungal agent

ABSTRACT

A correct viable cell number of a microorganism in a biological tissue is measured by cultivating a fragment of the biological tissue such as skin infected with the microorganism and administered with an antimicrobial agent, in a medium containing a phospholipid, a nonionic surfactant, or both of the phospholipid and the nonionic surfactant.

TECHNICAL FIELD

[0001] The present invention relates to a method for quantitating amicroorganism in a biological tissue and a method for evaluating anantimicrobial agent based on the use of the same.

BACKGROUND ART

[0002] The medical treatment for the microbial infectious disease is oneof the great tasks even in these days in which a large number ofantimicrobial drugs have appeared. One of the causes of such a situationis the fact that the sensitivity of the microorganism to theantimicrobial agent always changes due to some affairs such as theacquisition of resistance. Another cause is the fact that certainmicroorganisms, which are not exterminated by the antimicrobial agent,still exist. As for the causes as described above, the representativecases of the latter involve fungi which cause the dermatophytosis. Inthe case of the latter, the cause of the almost unsuccessfulextermination resides in the fact that there is no evaluation systemwhich makes it possible to screen an agent that is effective upon actualadministration to a living body.

[0003] The reason, why there is no effective screening system for theantifungal agent as described above, includes the following fact.Usually, the screening for the antifungal agent involves the in vitroscreening in which the influence exerted on the growth of fungi isobserved on a medium, and the in vivo screening in which an agent isadministered to an animal to be infected in vivo, a segment or fragmentof a biological tissue therefrom is transplanted to a medium, and theviable cell number of fungi proliferated thereby is counted to estimatethe number of fungi surviving in the biological tissue. It is assumedthat the in vitro screening is indicative of the antifungal activity ofthe agent, while the in vivo screening is representative of the effectbased on those ranging to the absorption and metabolism characteristicsof the living body. However, in the case of the in vivo screening forthe antifungal agent or the like, the agent remains at theadministration site, and the growth of fungi is inhibited thereby. As aresult, the number is counted as if the viable cell number is decreased,in which no appropriate screening is performed for the agent. Any means,which inactivates the agent remaining as described above, has not beenknown yet until the present time. That is, although it is demanded torealize a means for inactivating the agent remaining in the tissue inorder to measure a correct viable cell number in the tissue, such ameans has not been obtained yet. Therefore, it is affirmed that thisfact impedes the development of effective antifungal agents for theskin.

DISCLOSURE OF THE INVENTION

[0004] The present invention has been made taking the foregoingcircumstances into consideration. An object of the present invention isto provide a means for measuring a correct viable cell number in atissue of a living body.

[0005] As a result of diligent and repeated investigations and effortsperformed by the present inventors in order to seek for a means formeasuring a correct viable cell number in a tissue of a living body, ithas been found out that the influence of an agent remaining in a tissuecan be eliminated by cultivating a segment or fragment of the biologicaltissue in a medium containing a phospholipid and/or a nonionicsurfactant, and thus the invention has been completed. That is, thepresent invention relates to the technique as specified below.

[0006] (1) A method for quantitating a microorganism in a biologicaltissue, comprising cultivating a fragment of the biological tissueinfected with the microorganism and administered with an antimicrobialdrug, in a medium containing a phospholipid, a nonionic surfactant, orboth of the phospholipid and the nonionic surfactant, and detecting thegrown microorganism.

[0007] (2) The method for quantitating the microorganism in thebiological tissue according to (1), wherein the microorganism is afungus.

[0008] (3) The method for quantitating the microorganism in thebiological tissue according to (1) or (2), wherein the biological tissueis skin.

[0009] (4) A method for evaluating an antimicrobial agent, comprisingadministering the antimicrobial agent to a living body infected with amicroorganism, taking a fragment of a site of the administration of theagent thereafter, quantitating the microorganism in the fragment bymeans of the method for quantitating the microorganism as defined in anyone of (1) to (3), and using, as an index, a ratio of a microorganismamount obtained when the agent is administered to a microorganism amountobtained when the agent is not administered.

[0010] (5) The method for evaluating the antimicrobial agent accordingto (4), wherein the microorganism is a fungus.

[0011] (6) A method for evaluating an antifungal agent, comprisinginfecting skin of an animal with a fungus, treating the infected skinwith the antifungal agent thereafter, quantitating a viable cell numberof a site having been treated with the antifungal agent by means of themethod for quantitating the microorganism in the biological tissue asdefined in (2), and using the viable cell number as an index.

[0012] The present invention will be explained in detail below.

[0013] The method for quantitating the microorganism in the biologicaltissue according to the present invention is characterized in that thefragment of the biological tissue, which is infected with themicroorganism and which is administered with the antimicrobial agent, iscultivated in the medium containing the phospholipid, the nonionicsurfactant, or both of them.

[0014] The biological tissue to be used in the present invention is thetissue which is infected with the microorganism and which is treatedwith the histopathological agent. It is especially preferable to use atissue in which the agent is stored. The quantitating method of thepresent invention is useful in such a way that the influence of theagent, which would otherwise affect the situation of the microbialgrowth as described above, is canceled to count the correct microbialnumber, for the following reason. That is, the influence of the agent,which would be otherwise exerted on the microorganism, can be canceledby the phospholipid and/or the nonionic surfactant to be used for thequantitating method of the present invention. Even when each of thecomponents is used singly, the component exhibits an activity toinactivate the remaining agent. However, when the phospholipid and thenonionic surfactant are used in combination, a reliable activity isexhibited to inactivate the remaining agent. Therefore, any one of thecomponents can be used to inactivate the agent and quantitate the viablecell number in the biological tissue as well. However, in the presentinvention, it is especially preferable to use both of them.

[0015] Any phospholipid can be used in the present invention without anyspecial limitation provided that the phospholipid is commonly known.Those preferably exemplified may include, for example, lecithin,phosphatidylcholine, phosphatidylinositol, phosphatidylglycerol,phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, andlyso-phospholipids thereof. Among them, lecithin, which is availablemost easily, is especially preferred. The phospholipid as describedabove is preferably contained at a content of 0.1 to 10% by weight, andmore preferably 0.5 to 5% by weight in total amount with respect to thewhole quantity of the medium, for the following reason. That is, if thecontent is too small, it is impossible to obtain the activity toinactivate the agent in some cases. On the other hand, if the content istoo large, then the inactivating activity is not only saturated, butalso the phospholipid remains as solid in the medium to disturb theobservation in other cases.

[0016] Hydrophilic surfactants, each of which has an H.L.B(hydrophilic-lipophilic balance) of not less than 10, are preferablyusable as the nonionic surfactant to be used in the quantitating methodof the present invention. Especially, it is preferable to use thosehaving a form in which the polyoxyethylene group is added. The averagenumber of moles of added oxyethylene as described above is preferablynot less than 6 and not more than 100, and more preferably not less than10 and not more than 60, for the following reason. That is, if thehydrophilicity is either too high or too low, it is difficult toinactivate the remaining agent when the nonionic surfactant is combinedwith the phospholipid. Generally, the type of the nonionic surfactantusable in the quantitating method of the present invention may bepreferably exemplified as follows on the basis of the form of additionof the polyoxyethylene group. That is, those preferably exemplified mayinclude, for example, polyoxyethylene fatty acid ester, polyoxyethylenealkyl (alkenyl) ether, polyoxyethylene sorbitan fatty acid ester,polyoxyethylene cured castor oil, and polyoxyethylene glyceryl fattyacid ester. Among them, polyoxyethylene sorbitan fatty acid ester isespecially preferred, and oleic acid is especially preferred as thefatty acid of such a compound. That is, the nonionic surfactant, whichis used most preferably in the quantitating method of the presentinvention, is polyoxyethylene sorbitan oleic acid ester. Tween 80, whichis commercially available from Atlas Chemical Co., may be preferablyexemplified as a commercially available product of the nonionicsurfactant as described above. In the quantitating method of the presentinvention, the nonionic surfactant is preferably contained in the mediumat a content of 0.01 to 10% by weight, and more preferably 0.1 to 5% byweight in total amount with respect to the whole quantity of the medium,for the following reason. That is, if the content is too small, theeffect as described later on is not exhibited in some cases. On theother hand, if the content is too large, then the effect as describedlater on is saturated, and the growth of fungi or the like is inhibitedin other cases. This component acts singly, or it preferably actstogether with the phospholipid to exhibit the activity to inactivate theagent remaining in the fragment of the living body.

[0017] In the present invention, the biological tissue includes, forexample, skin, lungs (respiratory organs), and bowels (digestiveorgans). However, among them, the skin is preferred.

[0018] The microorganism, which is subjected to the quantitation inaccordance with the present invention, is not specifically limitedprovided that the biological tissue is infected with the microorganism.However, microorganisms, which cause the dermatological infection or theskin infectious disease, are preferred, and fungi are especiallypreferred, because the following tendency is widely known especially inthe medical treatment for the dermatophytosis or the dermatomycosis.That is, it is impossible to quantitate any correct viable cell numberof fungi surviving in the skin, because the agent is stored in the skinfor a long period of time. This consequently causes the difference inefficacy of the agent between the in vivo animal test and the clinicaltest. The microorganism, to which the present invention is applicable,specifically includes, for example, bacteria such as Helicobacterpylori, pathogenic Escherichia coli, and Staphylococcus aureus, andfungi such as Trichophyton mentagropytes, Trichophyton rubrum, Candidaalbicans, Cryptococcus neoformans, and Aspergillus fumigatus.

[0019] The fragment of the biological tissue infected with themicroorganism may be either a fragment which is taken from a tissue of aliving body naturally infected with the microorganism, or a fragmentwhich is taken from a tissue of a living body after infecting the livingbody with the microorganism by means of a method suitable for themicroorganism.

[0020] The antimicrobial agent can be evaluated by utilizing the methodfor quantitating the microorganism in the biological tissue according tothe present invention. That is, when the method of the present inventionis used, the viable cell number can be quantitated without beingaffected by the stored agent. Therefore, it is possible to obtain, evenin the in vivo test, data which is well correlated with results of theclinical test. Accordingly, it is possible to allow agents having lowefficacy to drop out without testing them in the clinical test. It isnoted that when such an operation is performed with an experimentalinfected animal, an efficacy assay test may be constituted. When aninfected and medically treated site, which is obtained from an infectedperson such as a patient, is subjected to the biopsy and thequantitation, it is possible to monitor the medical treatment process.When the biological tissue is taken from an animal or a patient asdescribed above, it is preferable that about 3 to 20 fragments, each ofwhich has a size of about 1-10 mm×1-10 mm, are uniformly taken from acertain site. As for the medium to which the fragment is transplanted,any medium can be used without any special limitation provided that themedium is known as a growth medium for the objective microorganism. Inthe case of the microorganism such as fungi, those preferablyexemplified may include, for example, Sabourand medium, Sabourandmodified medium, and RPMI medium.

[0021] An embodiment of the method for evaluating the agent is describedbelow.

[0022] (1) An experimental animal is infected with the microorganism toprepare an infected animal. In this process, a preferred form of theinfection is local infection. In the case of the therapeutic agent forthe dermatophytosis, the following procedure may be adopted. That is,the back of the experimental animal such as guinea pig is previouslysubjected to hair shaving, or bottoms of feet are used in an untreatedstate. Conidia are picked from a preliminarily cultivated fungus toprepare infectious solutions having uniform concentrations thereof. Whenthe infectious solutions are percutaneously administered, for example,by means of the closed patch, it is possible to prepare an animal modelinfected with the dermatophytes.

[0023] (2) The infected site of the infected animal is treated byadministering the agent. Specifically, the treatment method includes,for example, methods of application of the agent solution, oraladministration, and intravenous injection.

[0024] (3) A fragment of the biological tissue is taken from theagent-administered site of the infected animal. In the case of the skin,the skin is taken out by means of the excision.

[0025] (4) The fragment of the biological tissue is inoculated. Amedium, which contains the phospholipid, the nonionic surfactant, orboth of them, is previously prepared. The fragment of the biologicaltissue is inoculated to the medium to perform the cultivation. Thecultivation is usually performed under a condition including, forexample, a temperature suitable for the growth of the microorganism.

[0026] (5) After the cultivation, the microbial number is counted, orthe size of a formed colony is measured for the microorganism grown fromthe fragment of the biological tissue to use an obtained result as anindex of the number of surviving microbes or the viable cell number. Inthis process, the following procedure is also advantageous. That is,plates, to which several solutions having different concentrations ofconidia are inoculated, are separately prepared to make comparisontherewith so that the number of surviving cells itself is estimated.Further, an infected animal, which is not treated with the agent, isprepared to make comparison therewith. Accordingly, it is possible toevaluate the antimicrobial effect of the agent. Specifically, it ispossible to use, as an index, the survival rate, i.e., the ratio of amicroorganism amount obtained when the agent is administered to amicroorganism amount obtained when the agent is not administered.

[0027] In a more preferred embodiment, the behavior is confirmed with amedium which contains neither phospholipid nor nonionic surfactant, forthe following reason. That is, when such a control is provided forcomparison, it is possible to know whether or not the phospholipidand/or the nonionic surfactant adequately inactivates the agent.

BRIEF DESCRIPTION OF THE DRAWING

[0028]FIG. 1 shows a way of excision of foot skin in Example 2.

BEST MODE FOR CARRYING OUT THE INVENTION

[0029] The present invention will be more specifically explained belowas exemplified by Examples. However, it goes without saying that thepresent invention is not limited to only Examples.

EXAMPLE 1

[0030] MIC (minimum inhibitory concentration) was measured by means ofthe microdilution method by using 96-well plates to investigate theinactivation of respective agents caused by lecithin and Tween 80. Thefollowing four media were used. That is, a Sabouraud liquid medium (SDB)as a base medium, a medium obtained by adding 0.7% Tween 80 to SDB, amedium obtained by adding 1% lecithin to SDB, and a medium obtained byadding 0.7% Tween 80 and 1% lecithin to SDB were used. The followingagents were used. That is, lanoconazole, bifonazole, and terbinafinewere used. The following test strain was used. That is, Trichophytonmentagropytes TIMM 2789 (available from Institute of Medical Mycology ofTeikyo University), which is generally used for the animal infectionexperiment, was used. The cultivation was performed at 28° C. for 7days, while the microbial concentration upon the inoculation was about10⁴ conidia/ml. After the cultivation, it was regarded that MIC (μg/ml)was represented by the minimum agent concentration of the plate on whichno microbial growth was observed by visual observation.

[0031] Obtained results are shown in Table 1. According to Table 1, itis clarified that any one of the antifungal agents is inactivated bylecithin as the phospholipid and/or Tween 80 as the nonionic surfactant.Further, it is appreciated that the agents can be inactivated morereliably in a form in which both of the phospholipid and the anionicsurfactant are contained, as compared with a form in which each of themis used singly. TABLE 1 Additive Bifonazole Lanoconazole Terbinafinenone 1.56 ≦0.005 ≦0.005 Tween 80 >25 0.312 0.312 Lecithin >25 0.0780.078

EXAMPLE 2

[0032] The effects of the method for quantitating the microorganism inthe biological tissue and the method for evaluating the antifungal agentaccording to the present invention were confirmed by using an animalmodel infected with the dermatophytes. Female Hartley guinea pigs weregrouped into several groups each including 5 individuals to prepare afoot trichophytosis model by using a method modified by Uchida et al.originating from an original method of Fujita et al. (Fujita, S., andMatsuyama, T., 1978, “Experimental tinea pedis induced by non-abrasiveinoculation of Trichophyton mentagropytes arthrospores on the plantarpart of a guinea pig foot”, J. Med. Vet. Mycol., 25, 202-213; andUchida, K. & Yamaguchi, H., 1996, “Preclinical therapeutic evaluation ofagents for treating dermatophytosis”, Jpn. J. Med. Mycol., 37, 199-205).That is, Trichophyton mentagropytes TIMM 2789 strain was used to adjustthe concentration to 2×10⁷ conidia/ml. Pad-equipped adhesive plasters,in which 100 μl of the suspension of conidia was allowed to permeateinto lint cotton portions, were fixed to bottoms of feet of right andleft hind legs of guinea pigs by using surgical tapes. The adhesiveplasters were removed on the 7th day after the fixation. The medicaltreatment was started on the 28th day after the infection. Acommercially available formulation of 1% lanoconazole cream (Astat(trade name)), a commercially available formulation of bifonazole(Mycospor), or a commercially available formulation of terbinafine(Lamisil) was externally applied in an amount of 0.1 g once a day for 3days. It is noted that the solution used for the test containedpolyethylene glycol 400 and ethanol (75:25 vol/vol).

[0033] Skin pieces (1 mm×2 mm) were excised on the 7th day and on the14th day after the final treatment, while being divided into twovertically and into ten laterally (divided into twenty in total, seeFIG. 1). Ten slices of those divided into two vertically were inoculatedto a Sabouraud agar medium (SDA) containing 1% lecithin and 0.7% Tween80, and remaining ten slices were inoculated to SDA to observe thepresence or absence of the fungus on both of the media. (Antibioticswere added to SDA used in accordance with the ordinary method.)

[0034] Obtained results are shown in Table 2. According to Table 2, itis appreciated that the agent remaining in the skin behaves as anobstacle for the screening in the in vivo test for the antifungal agent,and that the obstacle can be eliminated by using the quantitating methodof the present invention. TABLE 2 Number of positive cases SDA mediumonly 7th day after treatment no treatment control 10/10 Astat cream 0/10 Mycospor cream 10/10 Lamisil cream  0/10 14th day after treatmentno treatment control 10/10 Astat cream  0/10 Mycospor cream 10/10Lamisil cream  0/10 Added with lecithin and Tween 80 7th day aftertreatment no treatment control 10/10 Astat cream  5/10 Mycospor cream10/10 Lamisil cream  3/10 14th day after treatment no treatment control10/10 Astat cream  0/10 Mycospor cream 10/10 Lamisil cream  3/10

INDUSTRIAL APPLICABILITY

[0035] According to the present invention, it is possible to provide themeans for measuring the correct viable cell number in a tissue of aliving body.

1. A method for quantitating a microorganism in a biological tissue,comprising cultivating a fragment of the biological tissue infected withthe microorganism and administered with an antimicrobial drug, in amedium containing a phospholipid, a nonionic surfactant, or both of thephospholipid and the nonionic surfactant, and detecting the grownmicroorganism.
 2. The method for quantitating the microorganism in thebiological tissue according to claim 1, wherein the microorganism is afungus.
 3. The method for quantitating the microorganism in thebiological tissue according to claim 1 or 2, wherein the biologicaltissue is skin.
 4. A method for evaluating an antimicrobial agent,comprising administering the antimicrobial agent to a living bodyinfected with a microorganism, taking a fragment of a site of theadministration of the agent thereafter, quantitating the microorganismin the fragment by means of the method for quantitating themicroorganism as defined in any one of claims 1 to 3, and using, as anindex, a ratio of a microorganism amount obtained when the agent isadministered to a microorganism amount obtained when the agent is notadministered.
 5. The method for evaluating the antimicrobial agentaccording to claim 4, wherein the microorganism is a fungus.
 6. A methodfor evaluating an antifungal agent, comprising infecting skin of ananimal with a fungus, treating the infected skin with the antifungalagent thereafter, quantitating a viable cell number of a site havingbeen treated with the antifungal agent by means of the method forquantitating the microorganism in the biological tissue as defined inclaim 2, and using the viable cell number as an index.